The present invention relates to the field of equilibrium dialysis devices. More particularly, the present invention describes a micro-equilibrium dialysis apparatus utilizing any number of dialysis membranes vertically inserted through a dialysis block containing any number of sample wells, such that both the donating and receiving sides of all wells can be accessed from the top of the apparatus at any time.
Equilibrium dialysis is a procedure for measuring the concentration of free, relatively small molecules in a sample. The procedure was originally designed to study the quantitative aspects of immunity reactions and, over the years, the procedure has been employed primarily in immunological studies. See, e.g., J. Marrack and F. C. Smith, Brit. J. Exptl. Path., 13, 394 (1932), F. Haurowitz and F. Breirl, Z. Physiol. Chem., 214, 111 (1933), H. N. Eisen and F. Karush, J. Am. Chem. Soc., 71, 363 (1949), and D. N. Weir, Editor, xe2x80x9cHandbook of Experimental Immunologyxe2x80x9d, Second Edition, Blackwell Scientific Publications, Oxford, 1973, pp. 16.1-16.21 . Furthermore, equilibrium dialysis has been considered an ideal approach to study binding of small molecules or ions (ligants) to macromolecules (proteins) and such studies have been very important in many fields, including biochemistry and pharmacology.
In principle, equilibrium dialysis can be employed to provide a sample from which one can determine the concentration of relatively small molecules; the only requirement is that the material to be measured must pass freely through a semi-permeable membrane. Once equilibrium has been achieved, since the concentration of the freely movable material is the same on both sides of the membrane, it is only necessary to analyze the dialysate for the desired material.
Historically, equilibrium dialysis applications, such as binding and affinity studies, have been slow, difficult and costly to perform. One of the best currently available devices for equilibrium dialysis, for example, only allows for a maximum of twenty samples to be tested simultaneously per device and requires expensive equipment which is often difficult to use and timely to assemble. The device also is not adaptable to standard 96-well pipetting equipment nor does it easily lend itself to automation through the use of robotic systems. Additionally, the orientation of the equipment results in difficulty accessing or removing independent samples during dialysis.
This currently available device utilizes oval or circular dialysis membranes which are placed on top of a hemispherical or cylindrical lower half of the testing cell and over which is joined the corresponding top half of the cell. The samples are inserted into the cells using a syringe with a blunt nose needle. Up to five cells can be stacked together and once assembled and filled, the stack is rotated continuously on the axis perpendicular to the dialysis membranes on a spit-like mechanism. Four rows of cells can be placed on top of one another in the dialyzer such that a maximum of twenty cells can be utilized at one time. The temperature of the dialyzer can be varied between xe2x88x9210 and 65xc2x0 C. using a water bath or a coldroom, and the cells can be rotated at adjustable speeds between 5 and 30 RPM.
One problem with this spatial orientation is that it requires continuous rotation of the test cells so that the samples remain in constant contact with the dialysis membrane so as to avoid concentration polarization. Additionally, this orientation, in a system which is scaled down, such as in the case of the present invention, would result in other problems such as air bubbles which become trapped against the dialysis membrane and slow or prohibit dialysis and the inability to maintain the surface area to volume ratio of the sample as the volume of sample to be tested changes.
It is, therefore, an object of the present invention to provide for an improved device for conducting equilibrium dialysis assays.
Another object of the present invention is to greatly reduce the cost of conducting equilibrium dialysis assays.
Another object of the present invention is to improve upon current methods of conducting equilibrium dialysis assays by providing for vertical placement of dialysis membranes though the well so that both sides of the well are accessible at all times.
Another object of the present invention is to utilize a material for the construction of the dialysis block such that non-specific binding of the samples tested to the surface of the block is minimized.
Broadly, the present invention discloses a dialysis block comprising a top and bottom surface, containing one or more wells separated by a dialysis membrane into sections accessible from the top of the device, in which wells a testing substance may be placed. In one specific embodiment, a plurality of prefabricated well-membrane bodies are placed into wells formed into the dialysis block. The well-membrane bodies are preferably formed as a singular body, by any of a variety of methods such as by injection molding. In another specific embodiment, each well is divisible by a gap passing along a vertical plane perpendicular to the top and bottom planes of the block and passing through the entire depth of the well. A dialysis membrane is placed in the gap, dividing the well into two, for the sample and dialysis buffer respectively. The gap preferably is of a depth larger than the depth of any well formed and preferably of a diameter larger than that of such well so as to minimize leaking of the test substance between the two sides of the well.
The block may be made of any of a variety of shapes, sizes or materials. The material of the block is preferably a material that will minimize non-specific binding of the samples to be tested. Suitable materials include some type of PTFE (polytetrafluoroethylene or Teflon(copyright)). Any number of wells may be used. Also, wells of any depth and diameter may be formed. In one suitable implementation, the number of wells is ninety-six, arranged in an 8xc3x9712 array, of such predetermined spacing and dimensions as to make the wells accessible to and compatible with all standard 96-well format laboratory supplies and instruments. Preferably, the wells are formed by drilling holes into the dialysis block.
Preferably, the gaps in the wells through which the dialysis membrane may be placed are formed by using two or more bars to form the body of the block. The wells are typically formed so that they overlap with, and are split by, the gaps formed between adjacent bars. When the wells are formed in this manner, the planar gaps between adjacent bars which are perpendicular to the top and bottom surfaces of the block, provide gaps of necessary thickness and depth for the placement of the dialysis membrane.
Preferably, the bars are formed by cutting larger blocks to predetermined dimensions and then milling the individual bars to make them flat. In one preferred embodiment, nine such bars are used to provide sufficient spacing for an array of ninety-six wells arranged in eight rows and twelve columns. In such an embodiment, the eight membranes are placed between each of the nine rows of bars and divide each of the ninety-six wells into even halves along a diameter of the well. The nine rows of bars are then held together by any method, preferably clamping, in order to prevent leakage of the sample tested.
Optionally, two or more alignment pins, of a length sufficient for all bars to rest on the pins, are inserted through holes formed in each of the two sides of the bars added. The bars are constructed to slide on the pins in a horizontal plane relative to one another to simplify set-up and usage.
One advantage of the present invention is to provide for the ability to conduct much larger numbers of assays in a given time frame, allowing an investigator to conduct a wider range of experiments than previously considered possible. For example, using a preferred embodiment of the present invention containing sample wells arranged in a 96-well array so as to be compatible with standard 96-well format laboratory supplies and instruments, and combining eight such arrays in parallel, an investigator could easily and cheaply conduct over one thousand assays in the same time previously needed to conduct twenty with a device embodying the prior art. Additionally, using a device embodying the prior art, up to four hours of investigator time were required for set-up, clean-up and collection of results from one twenty-sample test group. Using the preferred embodiment of the present invention described above, and combining eleven such devices in parallel, the time required for an investigator to perform the same tasks to conduct over 1000 dialysis assays is less than three hours.
Another advantage of the preferred embodiment is its compatibility with standard 96-well pipetting equipment and the ability to easily automate the processing of donating and receiving samples of the dialysis assay using readily available robotic systems. Such an embodiment allows the investigator to analyze a much larger number of samples, time points, or replicates in the same experiment than previously possible using the prior art. Furthermore, by varying the dimensions of the wells and their location relative to one another, arrays of various sizes and dimensions can be formed so as to make the wells compatible with other and future laboratory supplies and equipment.
Additionally, any embodiment incorporating the present invention can be agitated by shaking or rotating the device in controlled temperature environments, thereby decreasing the time required to achieve equilibrium or alternatively stabilizing the compounds being studied.
Other advantages of the present invention, resulting from the placement of the dialysis membranes vertically through the wells rather than on top of the wells, include: (1) the ability to dispense to and/or sample from either or both the sample and dialysate sides from the top of the apparatus at any time during the experiment without taking the apparatus apart and stopping the assay; (2) the elimination of problems associated with trapped air pockets such as an increase in the time required for reactions to reach equilibrium; and (3) the ability to add or remove specific samples independently from the others. Furthermore, this orientation of the dialysis membrane maximizes the surface area to volume ratio and maintains the surface area to volume ratio as the volume of the reaction is increased, thereby decreasing the time required for reactions to reach equilibrium.
One additional advantage of the preferred embodiment of the present invention is a significant reduction in the cost of the equipment required for conducting equilibrium dialysis assays. The preferred embodiment of the present invention is easy and inexpensive to construct, quick and easy to assemble, use, disassemble and clean, and, with the exception of the dialysis membranes, reusable.